Severe hypercholesterolemia,impaired fat tolerance,and advanced atherosclerosis in mice lacking both low density lipoprotein receptor-related protein 5 and apolipoprotein E

LDL receptor-related protein 5 (LRP5) plays multiple roles, including embryonic development and bone accrual development. Recently, we demonstrated that LRP5 is also required for normal cholesterol metabolism and glucose-induced insulin secretion. To further define the role of LRP5 in the lipoprotein metabolism, we compared plasma lipoproteins in mice lacking LRP5, apolipoprotein E (apoE), or both (apoE;LRP5 double knockout). On a normal chow diet, the apoE;LRP5 double knockout mice (older than 4 months of age) had approximately 60% higher plasma cholesterol levels compared with the age-matched apoE knockout mice. In contrast, LRP5 deficiency alone had no significant effects on the plasma cholesterol levels. High performance liquid chromatography analysis of plasma lipoproteins revealed that cholesterol levels in the very low density lipoprotein and low density lipoprotein fractions were markedly increased in the apoE;LRP5 double knockout mice. There were no apparent differences in the pattern of apoproteins between the apoE knockout mice and the apoE;LRP5 double knockout mice. The plasma clearance of intragastrically loaded triglyceride was markedly impaired by LRP5 deficiency. The atherosclerotic lesions of the apoE;LRP5 double knockout mice aged 6 months were approximately 3-fold greater than those in the age-matched apoE-knockout mice. Furthermore, histological examination revealed highly advanced atherosclerosis, with remarkable accumulation of foam cells and destruction of the internal elastic lamina in the apoE;LRP5 double knockout mice. These data suggest that LRP5 mediates both apoE-dependent and apoE-independent catabolism of plasma lipoproteins.     

Identification of novel peptide agonists from a random peptide library for a 5-oxo-ETE receptor, a receptor for bioactive lipids

A combinatorial peptide library contains an enormous combination of amino acid sequences and drug candidates, but an effective screening strategy to identify a variety of bioactive peptides has yet to be established. In this article, a random hexapeptide library was screened to identify novel peptide ligands for a 5-oxo-ETE receptor (OXER), which is a G-protein-coupled receptor for bioactive lipids, by using an OXER-Gi1alpha fusion protein. We successfully identified 2 hexapeptides-Ac-HMQLYF-NH2 and Ac-HMWLYF-NH(2)-that exhibited agonistic activity. Although the corresponding affinities were relatively low (EC50 values of 146 and 6.7 microM, respectively), the activities were confirmed by other independent cell-based assay methods, namely, intracellular calcium mobilization and cell chemotaxis. This study demonstrates that a combinatorial peptide library may be screened using a [35S]GTPgammaS binding assay with G-protein-coupled receptor (GPCR)-Galpha fusion proteins, in general, and that of peptide ligands can be obtained even for nonpeptide receptors.     

Liposomes destabilize tRNA during heat stress

Biomembranes play an important role in cellular response to heat stress. In this study, we focus on the interaction between liposomes and tRNA. Upon heat treatment we determined circular dichroism spectra of tRNA in presence of liposomes prepared from POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and cholesterol (Ch). To compare thermal stability, midpoint temperature (T_m) of tRNA was calculated from normalized θ₂₀₈. Addition of POPC/Ch liposomes decreased the T_m value of tRNA from 48°C to 38°C. We conclude that POPC/Ch liposomes interact with tRNA and destabilize its conformation under heat stress.     

Cry1Aa binding to the cadherin receptor does not require conserved amino acid sequences in the domain II loops

Characterizing the binding mechanism of Bt (Bacillus thuringiensis) Cry toxin to the cadherin receptor is indispensable to understanding the specific insecticidal activity of this toxin. To this end, we constructed 30 loop mutants by randomly inserting four serial amino acids covering all four receptor binding loops (loops α8, 1, 2 and 3) and analysed their binding affinities for Bombyx mori cadherin receptors via Biacore. High binding affinities were confirmed for all 30 mutants containing loop sequences that differed from those of wild-type. Insecticidal activities were confirmed in at least one mutant from loops 1, 2 and 3, suggesting that there is no critical amino acid sequence for the binding of the four loops to BtR175. When two mutations at different loops were integrated into one molecule, no reduction in binding affinity was observed compared with wild-type sequences. Based on these results, we discussed the binding mechanism of Cry toxin to cadherin protein.     

Cloning,heterologous expression,and localization of a novel crystal protein gene fromBacillus thuringiensis serovarjaponensis strain Buibui toxic to scarabaeid insects

RecombinantEscherichia coli strains harboring pAG1, pAG2, pKBB100, and pKBB101 were cloned by using antiserum constructed against 130-kDa crystal protein antigen ofBacillus thuringiensis serovarjaponensis strain Buibui. DNAs in the recombinant strains hybridized to the 26-base oligonucleotide probe corresponding to N-terminal amino acids of the 130-kDa crystal protein of strain Buibui. Cultures of the recombinant strains were toxic to larvae of the cupreous chafer,anomala cuprea. Furthermore, the production of the 130-kDa polypeptide was demonstrated in the cells harboring pAG1 and pAG2 by immunoblot analysis with antiserum against the 130-kDa crystal protein. Southern hybridization analysis showed that the 130-kDa crystal protein gene is located on the chromosomal DNA of strain Buibui. On the other hand, DNA probes derived fromcryIA(a) andcryIIIA genes did not hybridize to the DNA of strain Buibui.     

Some Characteristics of Membrane-Associated Protein Kinase in Lemna paucicostata

A protein kinase which phosphorylates histone was isolated fromthe endoplasmic reticulum-rich fractions of Lemna paucicostata.The enzyme could be solubilized by sonication, and its molecularweight was estimated as 220,000 by Sephacryl S-300 gel filtration.The optimum pH for enzyme activity was 9.0–9.5 and theactivity was stimulated by Co^2$, Mg^2$ and Mn^2$. Substrate proteinswhich might be phosphorylated by this protein kinase were alsodetected in microsomal fractions of Lemna plants. ¹ Present address: Advanced Research Laboratory, HITACHI LTD.,Kokubunji, Tokyo 185, Japan.